Jones D et al. (1992) Worm Breeder's Gazette "A Ubiquitin Fusion Gene from C. elegans"

Candido EPM, Jones D

[Worm Breeder's Gazette, 1992]

Eukaryotes in general have three types of ubiquitin genes: a polyubiquitin gene which encodes multiple tandem copies of ubiquitin transcribed as a polyprotein and two other genes which are fusions of ubiquitin with either a 52 aa or an 80aa ribosomal protein. The polyubiquitin gene from C. elegans was described a few years ago by Roger Graham (Mol. Cell. Biol. (1989)2: 268-271). Since then, we have been studying the regulation of this gene and searching for additional ubiquitin genes. Roger was able to isolate a clone for the 52 aa fusion using a protocol he described in WBG (1990) 11(2):69. A subclone of the ribosomal-protein encoding region was used to probe a YAC grid. A single positive was found, on chromosome III. The most notable aspect of the organization of this gene is the number and position of introns. Two introns occur in the ubiquitin portion of the gene. The position of the first is the same as that in four repeats of the polyubiquitin gene. (C. elegans is the only organism known to have introns in the polyubiquitin gene.) The second intron splits the two terminal glycine residues of ubiquitin. Neither of these intron positions coincides with those in any other 52 aa fusion gene [See Figure 1]. The sequence of a cDNA clone for this gene, isolated from Bob Barstead's lZAP library, shows that the message is trans-spliced. As an additional bonus, this gene has a recognizable TATA motif (unlike the poyubiquitin gene) which is 138 bp from the ATG start codon. We have produced and are currently injecting constructs of this gene. We expect it to be universally constitutive (since it encodes a ribosomal protein) and hope that the promoter may have some practical use. In addition, we may even find out why these ribosomal proteins are always made fused to ubiquitin and what the fate of the ubiquitin part of the fusion is. The search for the third ubiquitin gene continues

Winge P et al. (1994) Worm Breeder's Gazette "R-ras 1 and R-ras 2 (TC21) Homologs."

Gobel V, Friend S, Winge P, Fleming JT

[Worm Breeder's Gazette, 1994]

R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with bc1-2, the human homolog to ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.

Jones D et al. (1993) Worm Breeder's Gazette "The Activity of Ubiquitin Gene Promoters in Transgenic C. elegans"

Candido EPM, Jones D, Stringham-Durovic EG

[Worm Breeder's Gazette, 1993]

We have isolated two ubiquitin genes from C. elegans. One is the polyubiquitin locus described by Roger Graham et al. (Mol. Cell. Biol. (1989)9: 268-271) and the other gene encodes a fusion of ubiquitin with a ribosomal protein (WBG 12(3) 63). We reasoned that the latter gene should have a "universal" promoter since the ribosomal portion of the gene product is presumably required for ribosome function. To date, three constructs have been made and tested. It should be noted that the gene has two small introns, both in the ubiquitin coding region, and that the message is trans-spliced. The constructs were: [See Figure] No rolling transformants (stable or transient) were obtained with pPCUbil. In addition, we observed that injected animals laid several dead oocytes, suggesting that pPCUbi1 is a lethal construct. The fusion of the ribosomal protein with -Galactosidase may impair ribosome function or assembly. pPCUbi2 lacks all of the ubiquitin fusion coding sequence. In addition, 120 bp of the leader were removed, which eliminated the trans-splice acceptor sequence. This construct used an unc-54 ,rather than a ubiquitin, 3' end. pPCUbi2 showed minimal expression in five transgenic lines with only the occasional neuron staining. Probably removal of the trans-splice acceptor site knocked out transcription or processing. In pPCUbi3 ,we fused ubiquitin to -Galactosidase, but removed the ribosomal coding region. This fusion should be cleaved in vivo at the ubiquitin/NLS junction. This construct showed reasonable expression in the three extrachromosomal strains obtained. One strain expressed -galactosidase only in the intestine, another in the intestine and nerve ring, and the third stained extensively in neurons, including the nerve ring and other head ganglia, the ventral cord, and tail ganglia. Pharyngeal muscle and the pharyngeal-intestinal valve also stain in this strain. At high magnification, faint staining in the occasional cell of the lateral hypodermis and in uterine and/or vulval muscle was also detectable. Both of these latter patterns were not overwhelming however. While no expression was observed in the germ line or early embryos, a few pretzel staged embryos did stain. This pattern is still far more limited than we anticipated. Southern analysis suggests that the portion of ubiB encoding the ribosomal protein is present as a single copy in the genome; therefore, we expected that every cell and certainly embryos would constitutively express ubiB. It is possible that some important sequence elements involved in expression or in processing of the transcripts are still missing from the test fusion. We are currently trying more constructs to see if this is the case. Alternatively, we could be witnessing negative interactions between the roller plasmid, pRF4 ,and the ubiB-lacZ fusion, especially since the three strains obtained vary so much in their spatial expression patterns. Despite our initial optimism, the ubiquitin promoters have so far not demonstrated universal tissue expression. Previous analysis of the 5' sequences of the polyubiquitin gene ubiA, also failed to yield the expected tissue general pattern of expression (Stringham et al., Gene (1992) 113: 165-173). We conclude that the promoters of the ubiquitin genes in C. elegans are rather complicated and, unfortunately, are not neatly contained within a small region upstream from the start site. Supported by the Medical Research Council of Canada.

Emmons SW et al. (1978) Worm Breeder's Gazette "studies on the DNA of C elegans."

Emmons SW, Files JG, Rosenzweig WD

[Worm Breeder's Gazette, 1978]

During the past year we have initiated a study of the DNA of C. elegans with a number of long range objectives in mind. We would like to isolate the DNA from genetically defined regions of the genome in order to construct physical maps to go along with genetic maps. We would like to use isolated fragments of DNA as hybridization probes for studies of transcription. If the size of an isolated restriction fragment differs in two strains of the worm, e.g., Bristol and Bergerac, this size can be used as a phenotype to map the genetic location of the restriction fragment. In this way we hope to locate on the genetic map genes, such as the ribosomal genes, which can be physically isolated but in which mutations have not yet been identified. The size of restriction fragments can also be used as a sensitive method to search for changes in the primary structure of the DNA during development. Here we report our initial progress in these experiments. Isolation of nucleic acids We have found that worms can be completely dissolved and digested by proteinase K in 1% SDS at 65 C, allowing isolation of DNA of high molecular weight and poly-A-containing RNA which is active in an in vitro translation system. Worms, usually frozen in liquid N2 and ground with a mortar before melting, are taken up in .1M tris pH 8.5, . 05M EDTA, .2M NaCl, 1% SDS. (Freezing and grinding is probably not necessary.) Proteinase K (EM Laboratories, Inc.; available from Scientific Products) is added to 200 lambda/ml and the mixture is heated to 65 C for 15' with occasional gentle rocking to mix. During this time the mixture clears almost completely and all worm carcasses disappear. The highly viscous solution is then extracted three times with phenol and once with chloroform-isoamyl alcohol (24:1). DNA may be separated from RNA at this point by precipitating nucleic acids with ethanol and winding out the DNA. We further purify DNA from RNA by digestion with RNase followed by phenol extraction and ethanol precipitation. Unfortunately, worms, particularly adults, contain a particulate material (a polysaccharide?) which copurifies with DNA through organic extractions and ethanol precipitations. This material results in blue DNA solutions, and may be responsible for the indigestibility of some DNA preparations with restriction endonucleases. Much of this material can be removed by spinning the DNA solution at 20,000 rpm for half an hour, and we do this routinely. To obtain C. elegans DNA rigorously free of E. coli DNA, we allow hypochlorited eggs to hatch into M-9 buffer and then purify DNA from the hatched L1's. C. elegans ribosomal DNA DNA coding for 18s and 28s ribosomal RNA (rDNA) can be purified from the bulk of worm DNA as a high density (50% G+C) satellite on cesium chloride gradients (Sulston and Brenner, Genetics 77, 95-104, 1974). The ribosomal genes are tandemly repetitious, containing about 50 copies of each gene. Digestion of this rDNA-containing satellite with restriction endonucleases Bam HI or Sal I gives a single band, the ribosomal unit repeat of 6800 base pairs. The appearance of only one band indicates that the rDNA contains a rather homogeneous repeat, and is the only repetitive DNA in the satellite. This band hybridizes labelled ribosomal sequences at a level 50-100 fold greater than expected for a unique sequence. We have cloned the 6800 base pair Bam fragment thereby providing a probe for hybridization experiments. We have mapped a number of restriction sites within the ribosomal repeat unit. Total worm DNA is digested with the appropriate restriction enzyme(s), run on a 0.7% agarose gel and blotted onto nitrocellulose filter by the technique of Southern. The filter is hybridized to either iodinated 125I-rRNA or to nick-translated 32P- cloned rDNA to identify the restriction fragments containing rDNA. The restriction map is consistent with a homogeneous 6800 base pair unit repeat. Heterogeneous repeat units present in only one copy would not be seen in this analysis, but are currently being looked for. The approximate location of 18s and 28s genes and of spacer regions has been located on the map. Hybridization to Southern blots from heavily loaded gels of digested DNA show a few minor bands, which are presumably fragments from the ends of the tandem repeat, containing some overlap into non-ribosomal DNA. It is also possible that some minor bands are heterogeneous unit repeats. Cloning and characterization of these fragments is in progress. It is striking that the length of the unit repeat is smaller than that found in almost all other eukaryotes. This could be the result of small genes or of very short spacer regions. We have sized the C. elegans large and small rRNAs by electrophoresis of glyoxal-denatured RNA on agarose gels (McMaster and Carmichael, PNAS 74, 4835-4838, 1977) obtaining values of 1700 and 3350 nucleotides, smaller than other eukaryotic rRNAs. In an attempt to locate the ribosomal genes on the genetic map, and to study the inheritance of repetitive DNA, we have compared the restriction cutting patterns of N2 rDNA with those of other strains of C. elegans. Any difference in cutting pattern (most likely due to spacer differences) would be a phenotype, easily mappable. In a comparison of N2 with C. elegans var. Bergerac (J. Brun), rDNA cutting patterns were identical with each of 12 restriction enzymes used. Using several restriction enzymes, the rDNA cutting pattern was also the same with a strain of C. elegans isolated from the wild (D. Russel). rDNA from C. briggsae (B. Zuckerman) did give differences in restriction cutting patterns. The restriction map is similar to that of N2, although the unit repeat is 400 base pairs longer and a few cutting sites are added or deleted. Some fragments appear to be the same in both species. Unfortunately, attempts by us (as well as by Nigon and Dougherty, J. Exp. Zool. 112, 485-503, 1949) to cross C. elegans and C. briggsae have not succeeded. Work at establishing a genetic system for rDNA is continuing. Repeated sequences in C. elegans DNA Sulston and Brenner (Genetics 77, 95-104, 1974) have shown that the DNA of C. elegans contains repetitive components similar to those found in other eukaryotic organisms: namely, inverted repeats, highly repetitive sequences, moderately repetitive sequences, and uniclue sequences. We have undertaken the further characterization of these sequences. So far we have completed initial experiments on the inverted repeat sequences and the moderately repetitive sequences. Inverted repeats. We have studied inverted repeat sequences by electron microscopy and find them to be similar in every way to those found in other eukaryotic organisms. Inverted repeats are visualized by simply melting high molecular weight DNA and spreading it for electron microscopy using the formamide technique of Davis, Simon and Davidson (Methods in Embryology, Vol. XXI, p. 413, 1971). The inverted repeat sequences are then seen as double-stranded stems, or stems with loops at their end, sticking out from the largely single- stranded DNA. Of 37 inverted repeats visualized, those without terminal loops (78%) had stems with a number average length of 250 bp, and those with loops (22%) had stems with a number average length of 340 bp. The number average length of the loops was 800 bp. The inverted repeats appear to be located in clusters in the DNA. Clusters contain a few (3-6) inverted repeats separated by about a thousand base pairs, and clusters are separated from each other by 10 to more than 70 thousand base pairs of DNA containing no inverted repeats. Moderately repetitive sequences. Moderately repetitive sequences are sequences present in the range of 10 to 100 times in the genome. In most eukaryotic organisms about half of such sequences consist of short (300 bp) stretches of repetitive DNA surrounded by unique sequences, and a large fraction of the unique DNA is interspersed in this way, at about one thousand base pair intervals, with moderately repetitive sequences. A few organisms (Drosophila, Chironomous, honey bee, and Achyla--a water mold) lack these short, interspersed repetitive sequences. We have studied the interspersion pattern of repetitive DNA in C. elegans by reassociation kinetics and find that, like Drosophila and the others of the minority group, it appears to lack the highly interspersed component of repetitive DNA. We have compared the rate of reassociation of fragments averaging 300 (120-650) and 2000 (1000- 4000) base pairs in length, using hydroxyapatite binding to assay formation of double strands. DNA of L1's were used after labeling by nick-translation. For shearing, reassociation, and hydroxyapatite binding, the methods of Britten, Graham, and Neufeld (Methods in Enzymology, 29, 363, 1974) have been followed. Seventy-six percent of the 2000 base pair fragments reannealed at a rate expected for unique fragments of that length. This represents only a slight increase over the fraction of the 300 base pair fragments which carry repetitive DNA, an increase from 20% to 24%. This result is consistent with a lack of highly interspersed repetitive DNA. We are presently analyzing the length of moderately repetitive sequences by electron microscopy to determine whether any short repetitive sequences are present at all. Studies on cloned fragments of C. elegans DNA We have constructed a small clone bank of C. elegans restriction fragments. We have used the Bam restriction endonuclease and have inserted the fragments into the pBR313 driver plasmid. Recombinant DNA work with C. elegans DNA is at the P2-EK1 level. We will be happy to share recombinant plasmids. We are using the cloned fragments as hybridization probes to study restriction fragments in worm DNA. A restriction digest of whole- genome DNA is fractionated on an agarose gel and transferred to a millipore filter (a 'Southern transfer'). A plasmid containing a particular cloned fragment is then labeled by nick-translation and hybridized to the filter to reveal the fragments in the whole digest which carry sequences homologous to those of the cloned fragment. We have been analyzing the patterns produced in this way to answer a number of questions: 1. Are the patterns consistent with the arrangement of repetitive DNA determined by COT analysis; that is, do most fragments consist solely of unique sequences? 2. Are there any differences in the patterns given by germ-line and somatic-line DNAs? 3. Are there any differences in the patterns given by Bristol and Bergerac DNAs? These could be used for mapping. Are there any differences between C. elegans and C. briggsae patterns? 4. Can differences in these patterns be used to find cloned fragments that come from genetically defined regions, for example, from regions covered by deletions? By hybridizing 0.1 g of a plasmid nick-translated to more than 10+E7cpm/ g to a filter carrying a digest of a few micrograms of worm DNA we can detect unique fragments after an overnight exposure. We use flashed film and intensifying screens and expose the film at -70 C. We have found that hybridizations at low temperature (e.g., 32 C) in 50% formamide and without Denhardt's solution are convenient and work very well. Thirteen recombinant plasmids (with inserts ranging in size from 1, 000 to 18,000 base pairs) have been hybridized to filters carrying digests of DNA from N2 L4 hermaphrodites, N2 L1 hermaphrodites, Bergerac L1 hermaphrodites, and C. briggsae (mixed population). All hybridize to a fragment in N2 DNA equal in size to the cloned insert they carry, indicating that no rearrangements have taken place during cloning. Nine plasmids hybridize to several (up to 10) additional bands. Even most inserts of less than 2000 base pairs (5 out of 8) hybridize to more than one band. From the COT analysis described earlier we would expect 76% of such fragments to consist entirely of unique DNA. Whether these figures are inconsistent, and if so, why, remains to be seen. We have used L4 hermaphrodites as a source of 'germ line' DNA in these experiments. By comparing DNA from them to DNA from newly hatched L1's, which lack a gonad, we can search for restriction fragments present in the germ line but absent from the somatic line. No such fragments have been found; so far the L1 and L4 patterns are identical. We have also started to use DNA isolated from sperm nuclei (a gift from Michael Klass), which will allow a much more rigorous comparison of germ and somatic line sequences. (In addition we are hoping that a comparison of sperm and hermaphrodite DNAs will allow, by examining the relative intensities of bands, identification of fragments from the X-chromosome.) Comparison of the bands in Bergerac and Bristol DNA's shows that these DNAs are not identical. Five Bristol bands (including two of the cloned inserts) appear to have a different size in Bergerac; that is, they are missing in the Bergerac pattern and one new band is present. We would like to find out whether these differences are due to single base changes or to rearrangements. This degree of difference between these strains suggests that genetic mapping by restriction fragments is feasible. Comparison of the C. elegans patterns with those of C. briggsae shows (to our surprise) that these DNAs are highly diverged. None of the 13 cloned fragments is present unaltered in the briggsae genome, and in fact 9 hybridize to no fragment whatsoever in briggsae DNA. Since we expect (but have not checked) that the proteins of these ( almost indistinguishable) worms would be very similar, this raises the possibility that DNA sequences present in both species are coding sequences.